CN1277934C - Isometric primer extension method and kit for detection and quantification of specific nucleic acid - Google Patents
Isometric primer extension method and kit for detection and quantification of specific nucleic acid Download PDFInfo
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Abstract
A method for detecting and/or quantifying a target DNA or RNA present in a sample by an isometric primer extension method is disclosed. The method includes carrying out a primer extension reaction in the absence of a free nucleotide so that the primer extension reaction is stopped where the absent nucleotide would have been inserted. Thus, as the amount of incorporation of nucleotide on the primer extended product is detected or quantified by mass spectrometry, the amount of the target RNA or DNA in the sample is measured.
Description
The application be submit to June 8 calendar year 2001, denomination of invention divides an application for the Chinese patent application 01121115.6 of " being used for specific nucleic acid detects and quantitative different on the same group preface primer extension method and test kit thereof ".
Technical field
The present invention relates to use the method for different preface primer extension (iPE) method detection on the same group and quantitative specific DNA or RNA.
Background technology
Routine is used for detecting and the method for quantitative nucleic acid (as DNA and RNA) specific sequence comprises the Southern trace, and Northern analyzes and the RNA enzyme protection is analyzed and polymerase chain reaction (PCR) and other methods.If but consider the detection of certain specific RNA in the sample; just there are following defective in Northern trace and the analysis of RNA enzyme protection: the RNA sample that validity is limited, labour intensity is big, tolerance range is low, cost is high, sensitivity is low, needs are more, specific equipment, and will produce large number of biological danger and radwaste.Specifically, Northern trace and the analysis of RNA enzyme protection all need to finish analysis over 2-3 days.In addition, the Northern trace needs the RNA gel to run sample, RNA is transferred on the solid phase carrier, prepares probe and carries out hybridization.Susceptibility then needs 5 μ g samples to guarantee enough susceptibilitys.The theoretical basis of Northern trace is the hybridization between target nucleic acid and probe nucleic acid.In addition, the cost of each reaction is very high, and the amount of bio-hazard that produces and radwaste is also very many.
Similarly, the RNA enzyme protection analysis result that needed obtain being fit in 2-3 days.Testing sequence need prepare template DNA, prepares rna probe, carries out hybridization, enzymic digestion reaction and gel run sample.In order to obtain good result, need 1 μ g target RNA sample.The theoretical basis of RNA enzyme protection analysis is with hybridization and enzymic digestion reaction associating.Identical with Northern trace method, the cost that carries out this reaction is also very expensive.Also produce large number of biological danger and radwaste in addition.Table 1 has been listed the comparison of Northern trace, the analysis of RNA enzyme protection and many primer extension methods of the present invention various factors.
U.S. Patent No. 5,846,710 disclose the dna molecular that screens variation with primer extension method.But this patent and unexposed detection to target DNA in the sample or RNA.
U.S. Patent No. 5,994,079 discloses dna primer and specific RNA annealing formation RNA/DNA crossbred, and extends primer with reversed transcriptive enzyme.Detect crossbred with specific antibody to the RNA/DNA crossbred.But the method for target DNA or RNA in the unexposed test sample as described in the present invention of this patent.
This area needs a kind of method of simple, time-saving detection nucleic acid as can be seen, and this method sensitivity, cost are low, effective, and little to the influence of environment.The present invention of the following stated has satisfied these requirements.
Summary of the invention
The present invention has satisfied above-mentioned requirements.
The present invention relates to detect and the quantitative method of target nucleic acid in the sample, this method comprises:
(a) the preparation specificity is matched in one or more primers of target nucleic acid predetermined site;
(b) under highly rigorous condition, one or more primers and the target nucleic acid of (a) are annealed, obtain primer-target nucleic acid duplex at the target nucleic acid predetermined site;
(c) primer-nucleic acid duplex with (b) mixes with a mixture, and this mixture contains:
(1) the nonterminal daughter nucleus thuja acid of one or both or three types of free nonterminal daughter nucleus thuja acids and at least a type, it randomly be marked with detectable and
(2) have or do not have the terminator nucleotides of a certain type, one or both in this Nucleotide and (1) or three kinds of nonterminal daughter nucleus thuja acids are different;
(d) in suitable damping fluid, carry out primer extension by enzymatic or chemical reaction; With
(e) amount of marking signal on the Nucleotide of detection or quantitative primer extension, or
(f) detect with mass spectroscopy or the amount of the primer that quantitatively extends.
In aforesaid method, primer can be the multipolymer of a kind of nucleic acid primer, oligodeoxynucleotide, oligoribonucleotide or thymus nucleic acid and Yeast Nucleic Acid.Nucleic acid interested can be the multipolymer of thymus nucleic acid, Yeast Nucleic Acid or thymus nucleic acid and Yeast Nucleic Acid.
In a preferred embodiment, this method comprises with the combination that contains following nonterminal and terminator nucleotides:
(a) dATP, dCTP, dGTP, ddTTP or ddUTP,
(b) dATP, dCTP, dTTP or dUTP, ddGTP,
(c) dATP, dGTP, dTTP or dUTP, ddCTP,
(d) dCTP, dGTP, dTTP or dUTP, ddATP,
(e)dATP、dCTP、dGTP
(f) dATP, dCTP, dTTP or dUTP,
(g) dATP, dGTP, dTTP or dUTP or
(h) dCTP, dGTP, dTTP or dUTP.
Method of the present invention can be used at least a nonterminal daughter nucleus thuja acid that is marked with detectable.Detectable marker comprises enzyme or protein portion, radio isotope, fluorescence part or chemical group (as vitamin H).In addition, also available mass spectroscopy is carried out in the step of detection or quantivative approach.
The enzyme that is used for primer extension reaction of the present invention comprises the template dependent enzyme, as e. coli dna polymerase I or its Klenow fragment, T4DNA polysaccharase, T7DNA polysaccharase, thermus aquaticus archaeal dna polymerase, retrovirus reversed transcriptive enzyme or their combination.
Can to these and other purposes of the present invention more fully understanding be arranged from following description of the invention, incidental reference drawing and claim.
Description of drawings
Fig. 1-many primer extension reactions are with the mode chart of detection and quantitative specific DNA sequences.Fig. 2-detect and the quantitative mode chart of RNA with many primer extension methods.The figure cathetus is represented primer sequence, and lowercase a, g, c and t are the bases from primer extension, and some of them are marked with detectable marker.The commutative use of u and t.
Embodiment
The present invention relates to the detection of different preface primer extension (iPE) method and the quantitatively method of specificity nucleotide sequence on the same group.Generally, the present invention is with the target DNA in the sample or RNA and the hybridization of single or multiple Oligonucleolide primers.When having a kind of free nucleotide of, two or three type preliminary making, extend primer then with archaeal dna polymerase or reversed transcriptive enzyme.Extend continuously and at least aly in the four types required Nucleotide have neither part nor lot in reaction, or substituted by the terminator nucleotides of respective type (as dideoxy nucleotide).Signal by whether existing marker to produce on definite primer that extends detects and quantitative specific target nucleic acid then.Because the primer that extends separates with free nucleotide, the primer of extension can be used for the incorporation of evaluation of markers thing.
Extension will produce the nucleic acid of the primer extension of many equal length (different on the same group preface) corresponding to the primer of a certain position of target nucleic acid because sequence dependent mixed true quantitative Nucleotide.Quantitative these equal primers that extend, the just number or the quantity of energy accurate quantification target nucleic acid.If the copy of many target DNAs or RNA is arranged in the sample, the copy number that mixes the primer extension product of tape label Nucleotide also correspondingly increases, thereby produces stronger resultant signal.Therefore, the standard model of observed strength of signal in the unknown sample and known dna or RNA amount is compared, just can detect and/or quantitative sample in target DNA or RNA.In another embodiment, measure the quantity of these equal length primer extension nucleic acid, can detect or quantitative specific target nucleic acid with mass spectroscopy.
No a certain free nucleotide will make primer extension stop in the reaction buffer, former should insert the Nucleotide that lacks here.So just can obtain the primer extension product of discontinuous length.
Many conspicuous changes can be arranged in the scope of the invention.For example, in primer extension reaction, not only can lack one type Nucleotide, also can lack two or three types of nuclear thuja acid.Equally also can use various markers, be not limited only to the radioactive nuleus thuja acid, also can be fluorescence or enzymatic.
Here used " nucleic acid " or " Nucleotide " can be the multipolymers of thymus nucleic acid, Yeast Nucleic Acid or thymus nucleic acid and Yeast Nucleic Acid.Nucleic acid samples can be natural or synthetic.Nucleic acid samples can be the nucleic acid of natural origin, also can be from any organism.Some organisms that can be used for the inventive method are: plant, microorganism, virus, bird, vertebrates, invertebrates, Mammals, people, horse, dog, ox, cat, pig or sheep.Target nucleic acid can be natural, maybe can be that the body endoenzyme is anabolic, vitro enzyme is anabolic or non-enzymatic synthetic.
The sample that contains a kind of interested nucleic acid or multiple interested nucleic acid can contain the genomic dna from organism, their rna transcription thing or the cDNA that is prepared by these rna transcription things.The sample that contains a kind of interested nucleic acid or multiple interested nucleic acid also can contain the outer DNA of organism genome, their rna transcription thing or the cDNA that is prepared by these rna transcription things.Equally, this interested nucleic acid or these interested nucleic acid can be synthetic by the polymerase chain reaction.
Interested nucleic acid can comprise the non-natural nucleoside acid-like substance, as Hypoxanthine deoxyriboside or 7-denitrogenation-2 '-pancreatic desoxyribonuclease.It is stable that these analogues lose the dna double chain, and can be in double-stranded sample and primer annealing, and extension takes place, and need not complete disengaging latch.
Interested nucleic acid can comprise one or more moietys, makes can be from uncorporated reagent and/or the primer affine interested nucleic acid of isolating.For example, interested nucleic acid can contain vitamin H, makes to be incorporated into avidin or its analogue (being attached to solid phase carrier) by making vitamin H, affinely from uncorporated reagent and/or primer isolates interested nucleic acid.DNA that interested nucleotide sequence can comprise or RNA sequence, can by with the complementary sequence paired base (being attached to solid phase carrier) that is present in the nucleic acid, affinely from uncorporated reagent and/or primer isolate interested nucleic acid.Interested nucleic acid can be marked with detectable marker; This detectable can be different from any detectable that exists in the reagent or be attached to primer.
Here " normal oligodeoxynucleotide " or " normal base " are the wild-type or the previously known standard nucleotides bases of attempting to identify in this base site sudden change." standard nucleotides base " comprises any known base, can be wild-type or known mutant base (as long as this base is known and wishes to understand its variant).So as an example, normal base can be known wild-type base, and wants in this site searching sudden change.On the contrary, known base can be known mutant, then seeks the existence of wild-type base in this site.In addition, known normal base can be known mutant, and it is sought another mutant variation base.So method of the present invention can be applicable to be used for determining whether this site exists any known sequences of any other base variant.
The term here " primer " or " Oligonucleolide primers " refer to a kind of oligonucleotide, when being under the condition that exists the various factors such as Nucleotide and enzyme (as archaeal dna polymerase) and the temperature that is fit to and pH to allow synthetic and nucleic acid (template) chain complementary primer extension product, it has the ability as synthetic starting point.
Term " primer " also may be defined as any nucleic acid fragment that obtains from any source.For example, primer can produce by broken bigger nucleic acid fragment (as genomic dna, cDNA or the DNA that obtains from PCR).That is to say that the characteristic of primer is not subjected to how to obtain the restriction of this primer, no matter it is broken natural or nucleic acid or is obtained by the nucleic acid primer.In addition, primer can be oligodeoxyribonucleotide, oligodeoxyribonucleotide multipolymer, oligoribonucleotide, ribonucleoside acid copolymer or deoxynucleotide and ribonucleoside acid copolymer.Primer can be natural or synthetic.Oligonucleolide primers can be that the body endoenzyme is anabolic, vitro enzyme is anabolic or external non-enzymatic synthetic.Primer can be marked with detectable marker; This detectable marker can be different from any detectable that exists in the reagent or be attached to interested nucleic acid.In addition, the sequence of primer must be corresponding to the flanking sequence of interested specific position, the certified nucleotide base of this positioned adjacent or be its upstream.
In addition, primer must have with interested nucleic acid in Nucleotide hybridization or annealed ability.A kind of approach of desirable hybridization that realizes is to allow template dependency primer in fact or fully be complementary to known base sequence.
Oligonucleolide primers can comprise one or more compositions, and primer is connected in solid phase carrier, make can be from uncorporated reagent and/or interested nucleic acid the affine primer of isolating.These affine compositions comprise, but are not restricted to, and digitonin, magnetic bead and part are as protein ligands (comprising antibody).Preferable composition is a vitamin H.With in the experiment of vitamin H, the primer that contains vitamin H is incorporated into streptavidin (being attached to solid phase carrier) by vitamin H, can be from uncorporated reagent and/or interested nucleic acid the affine primer of isolating.The dna sequence dna that the Oligonucleolide primers sequence can comprise, by with nucleic acid in complementary sequence paired base (being attached to solid phase carrier), can never mix the affine primer of isolating in reagent and/or the interested nucleic acid.
Terminology used here " primer extension reaction " refers to can carry out template dependency nucleic acid building-up reactions under reaction conditions.Partly, can create the condition that template dependency primer extension reaction takes place by the existence of appropriate template dependent enzyme.Some template dependent enzymes that are fit to are archaeal dna polymerases.Archaeal dna polymerase can have many types.But archaeal dna polymerase must be that primer and template are dependent.For example can use Klenow fragment, T4DNA polysaccharase, T7DNA polysaccharase (" Sequenase "), thermus aquaticus archaeal dna polymerase or the retroviral reversed transcriptive enzyme of e. coli dna polymerase I or e. coli dna polymerase I.Available equally in some versions RNA polymerase is as T3 or t7 rna polymerase.In hybridization and extension, different polysaccharases must be with different conditions and different temperature ranges.
Amplification step of the present invention comprise be selected from the clone, transcribe, the amplification method of the isothermal duplication of polymerase chain reaction, ligase chain reaction (LCR), strand displacement amplification and ring mediation.
Terminology used here " primer extension chain " comprises, after primer is added into, and the chain opposite of formation with template in the duplex.Preferably, mix primer extension chain termination primer extension by terminator.
The nucleic acid that terminology used here " template " refers to comprises double-stranded DNA, single stranded DNA and RNA, or any their modification, and can be any length or sequence.
Terminology used here " terminator " or " chain terminator " refer to nucleic acid base, as A, G, C, T or U, or analogue, it can stop primer extension reaction effectively when mixing the primer extension chain opposite with template strand.Preferable terminator is a dideoxy nucleotide.Equally preferably, terminator or unmarked thing or underlined thing, but should be different from the marker of nonterminal.When terminology used here " terminator " or " chain terminator " were odd number, not meaning used was single nucleic acid molecule.The singulative of term " terminator " is meant the type of the Nucleotide, nucleic acid base or the nucleic acid analog that are used to analyze.For example, if terminator is ddA, then singulative refers to all ddA integral body, rather than the ddA unit molecule.In addition, " terminator " can be the shortage of particular type Nucleotide, like this can be by lacking the extension that specific nucleotide stops primer on the locus.For example, end at the opposite of " C " on the template strand, then in the primer extension reaction mixture, should comprise nonterminal base A, T and C, but " G " (with " C " complementation) can not be arranged if wish primer extension reaction.So, lacking complementary base and will stop primer extension reaction, its effect is the same with the effect that adds dideoxy terminator Nucleotide.
The nucleotide base that terminology used here " nonterminal " or " non-chain terminator " comprise does not stop primer extension reaction when mixing the primer extension chain.Preferably, a kind of nonterminal of mark at least in primer extension reaction.As used herein, when term " nonterminal " or " non-chain terminator " when being odd number, not meaning used is single nucleic acid molecule.The singulative of term " nonterminal " is meant the type of the Nucleotide, nucleic acid base or the nucleic acid analog that are used to analyze.For example, if terminator is G, then singulative is meant the integral body of all G, rather than refers to unit molecule G.
Terminology used here " mutant " or " sudden change " refer to that template strand is taken up an official post and why not are same as the base of wild-type or normal base.The sudden change that detects with the inventive method can be the sudden change of any kind, comprises single base mutation, insertion, disappearance or group translocation, if be affected near the base on the direct relative template of the base of 3 ' of annealed primer.
Terminology used here " marker " refers to any molecule that terminator or nonterminal daughter nucleus thuja acid provide detection signal that is connected in.Marker can be radioactive, chemiluminescent, protein ligands such as antibody, if when perhaps using fluorophor, the nonterminal daughter nucleus thuja acid base of each type can be with different fluorophors.The emmission spectrum of these fluorescent markers should be distinguishable.
In addition, determine to mix the method for the level of nucleotide base in primer extension product, available mass-spectrometric technique is measured, and in U.S. Patent No. 5,885, enumerates in 775, quotes as a reference here.
Here used phrase " highly rigorous hybridization conditions " refer to the nucleic acid hybridization condition as, but be not restricted to, use the 0.1XSSC wash conditions for 42 ℃.In conventional molecular biology manual, generally can find hybridization conditions, as people's such as Ausubel Current Protocols in Molecular Biology, Greene and Wiley, pub. (1994) quotes as a reference here.
Here used " thin-layer chromatography (TLC) " can carry out on the paper medium based on cellulose prods, but can be made up of any material that can allow the fine dispersion of molecule and form even one deck.This material comprises, but is not restricted to inorganics such as silica gel, aluminum oxide, diatomite or Magnesium Silicate q-agent.Organism comprises, but is not restricted to Mierocrystalline cellulose, polymeric amide or polyethylene powders.Thin-layer chromatography method general description is in chemical handbook, as be set forth in Freifelder, Physical Biochemistry-Applications to Biochemistry and Molecular Biology, second ed., publish (1982) by Freeman and Co., here quote as a reference, particularly the 8th chapter has been discussed especially thin-layer chromatography of chromatographic technique at the 229-232 page or leaf.
A kind of change to interested nucleic acid evaluation and/or quantivative approach is after carrying out extension under the suitable sex change condition, to separate the primer extension chain from nucleic acid interested.This sex change condition can comprise heating, alkaline condition, methane amide, urea, oxalic dialdehyde, enzyme and their combination.This sex change condition also can comprise with 2.0N NaOH to be handled.
It will be understood by those skilled in the art that terminator can be marked with the marker that is different from nonterminal, be used for distinguishing mixing of primer extension chain terminator or nonterminal.Be simplified illustration in the present patent application, terminator is exemplified as the Nucleotide that lacks particular type, but this explanation does not have any type of restriction to claim.The present invention also comprises isolabeling or unlabelled terminator, as long as the marker on the terminator is different from the marker on nonterminal.
Those skilled in the art are appreciated that equally part is known just can to design a kind of primer as long as template sequence has at least, and this primer can be incorporated into template strand and the combination of primer on template strand takes place.Those skilled in the art it can also be appreciated that method of the present invention can be by putting into practice with the some primers in or more the analysis tubes.
A characteristic of the inventive method is if several nonterminal is labeled equally, just can produce strong signal, because nonterminal of several marks mixes the signal that the primer extension chain has generation addition.When various terminators or nonterminal have special different marker, observe its signal and can improve tolerance range.
Another object of the present invention provides a kind of test kit and reagent, is used for as required quantitatively or non-quantitation ground, whether definite sample exists target nucleic acid quickly and accurately.Each composition of test kit can be contained in the suitable container separately.It is labelled that each container also can be discerned the form of its inclusion.In addition, bigger the storing in all containers that need composition that each packaged one-tenth can be placed in.And test kit is subsidiary to have explanation how to use the specification sheets of this test kit.This specification sheets can be imprinted on the test kit or be attached to wherein.
Following examples are used to illustrate the present invention, but claim is not had any restriction.
Embodiment
(Tels-tel, TX) method is extracted full RNA from rat brain with RNAzol B.Concentration with the full RNA of O.D.260nm absorbance measurement.The water of handling with the diethylpyrocarbonate (DEPC) of no RNA enzyme dilutes full RNA.The RNA diluent (as shown in table 2) of the various content of 5 μ l is placed each pipe, mix with 1 μ l synthetic Oligonucleolide primers 5 '-GTGGGAACCGTGTCA-3 ' (SEQ ID NO:1) then, this sequence is and rat brain specificity cDNA paired sequence (unpublished data).70 ℃ were heated this RNA-primer mixture 3 minutes, and cultivated 3 minutes on ice.After these are managed rapid turn, add RNA enzyme inhibitors, 0.5mM dATP, dGTP, 1 μ l dCTP α that 14 μ l contain the Tris-HCl damping fluid that final concentration is 20mM (pH 7.5), 15 units
32The reaction mixture of P and 100 MMVL-of unit reversed transcriptive enzymes, the beginning primer extension reaction.2 minutes termination reactions of 100 ℃ of reacting by heating pipes were carried out in reaction 20 minutes under 37 ℃.1 μ l reaction mixture is made thin-layer chromatography, and (TRIM USA Maryland) tells free dCTP α
32P.Use scintillometer (Beckman LS 5000) to measure the radioactivity of labeled primer then.Table 2 has shown the result.
Above-mentioned comprise in steps chemistry, operation and flow process be automatization or can automatization.Therefore, include preference pattern of the present invention in the robot workspace's of suitable programming operation, will significantly save cost and will increase the efficient of any diagnostic procedure (depending on the formality of specific nucleotide sequence in the nucleic acid that detection derives from biological sample or sequence difference).
Here all reference of quoting are all quoted as proof and are included in the specification sheets.
Table 1
The comparison that MPE method and Northern analyze and the RNA enzyme protection is analyzed
| Method | Institute's time-consuming | Experiment process | Susceptibility | Principle | Cost | Bio-hazard/atomic waste |
| Northern analyzes | 2-3 days | ● the RNA gel runs sample ● RNA shifts ● preparation probe ● hybridization | 5μg | Has only hybridization | High | High |
| The RNA enzyme protection is analyzed | 2-3 days | ● preparation template DNA ● preparation rna probe ● hybridization ● enzymic digestion ● gel runs sample | 1μg | Hybridization and enzymic digestion | High | High |
| MPE | 1 hour | ● primer extension | 1ng | Hybridization and specificity are extended | Low | Low |
Table 2
| RNA measures (ng) | The primer of mark (cpm) |
| 20 | 31552 |
| 10 | 29756 |
| 5 | 26066 |
| 2 | 16779 |
| 1 | 11156 |
| 0.5 | 6587 |
| 0 | 6703 |
Sequence table
＜110〉Wang Xiaobing
Morisawa Shinkatsu
＜120〉being used for specific nucleic acid detects and quantitative different on the same group preface primer extension method and test kit thereof
<130>900315
<140>
<141>
<150>US 60/209,987
<151>2000-06-08
<150>US
<151>2001-05-23
<160>9
<170>PatentIn Ver.2.1
<210>1
<211>15
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: synthetic oligonucleotide
<400>1
gtgggaaccg tgtca 15
<210>2
<211>70
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: synthetic oligonucleotide
<400>2
tgatcagcag gctgaaatcg tcgtggattg caacgacgcc gacgattctc gtcctttaag 60
gcgatagcat 70
<210>3
<211>16
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: synthetic oligonucleotide
<400>3
tcgtcggcgt cgttgc 16
<210>4
<211>12
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: synthetic oligonucleotide
<400>4
aaaggacgag aa 12
<210>5
<211>70
<212>RNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: synthetic oligonucleotide
<400>5
ugaucagcag gcugaaaucg ucguggauug caacgacgcc gacgauucuc guccuuuaag 60
gcgauagcau 70
<210>6
<211>8
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: synthetic oligonucleotide
<400>6
gcctgctg 8
<210>7
<211>9
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: synthetic oligonucleotide
<400>7
ccacgacga 9
<210>8
<211>8
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: synthetic oligonucleotide
<400>8
ttaaagga 8
<210>9
<211>5
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: synthetic oligonucleotide
<400>9
Claims (4)
1. one kind is detected or the quantitative method of at least a nucleic acid in the sample, it is characterized in that described method comprises:
(a) primer annealing is attached on the target nucleic acid;
(b) extend described primer, do not exist by the nonterminal daughter nucleus thuja acid that makes at least a A of being selected from, T, G and C to form different on the same group preface extension products, wherein, have at least a kind of in the described Nucleotide of existence by fluorescent mark; With
(c) detect mixing of fluorescently-labeled Nucleotide in this different on the same group preface extension products.
2. one kind is detected or the quantitative method of the target nucleic acid in the sample, it is characterized in that described method comprises:
(a) at least a specificity of preparation is matched in the primer in the predetermined position of described target nucleic acid;
At least a primer annealing described in (a) is attached on the described target nucleic acid, on this target nucleic acid predetermined position, obtains primer-nucleic acid duplex;
Primer-the nucleic acid duplex of (b) is mixed with nonterminal daughter nucleus thuja acid mixture, wherein, lack at least a Nucleotide that is selected from A, T, G or C that extends continuously at primer extension reaction of being used in this nonterminal daughter nucleus thuja acid mixture;
(d) carry out different on the same group preface primer extension reaction with enzyme or chemical reaction, wherein said primer extension with the mixture of ribonucleotides of (c) in the nonterminal daughter nucleus thuja acid complementary target nucleic acid Nucleotide place that lacks stop; With
(e) detection or the quantitatively amount of different on the same group preface primer extension product.
3. method as claimed in claim 2 is characterized in that, adopts size to select separation, UV absorption, dyeing or mass-spectrometric technique to detect or measure the amount of the primer extension product of described mark.
4. one kind is detected or the quantitative method of the target nucleic acid in the sample, it is characterized in that described method comprises:
(a) at least a primer annealing is attached on the target nucleic acid, wherein, described primer mark detectable marker;
(b) extend the primer of described mark, form different on the same group preface extension products by at least a existence the among A, the T, G or the C that are used in the continuous extension in the primer extension reaction;
(c) primer extension product of certification mark.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US20998700P | 2000-06-08 | 2000-06-08 | |
| US60/209,987 | 2000-06-08 | ||
| US09/862,417 | 2001-05-23 | ||
| US09/862,417 US6824980B2 (en) | 2000-06-08 | 2001-05-23 | Isometric primer extension method and kit for detection and quantification of specific nucleic acid |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011211156A Division CN1198145C (en) | 2000-06-08 | 2001-06-08 | Isometric primer extension method for specific nucleate test and quantitation and reagent kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1537955A CN1537955A (en) | 2004-10-20 |
| CN1277934C true CN1277934C (en) | 2006-10-04 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB011211156A Expired - Fee Related CN1198145C (en) | 2000-06-08 | 2001-06-08 | Isometric primer extension method for specific nucleate test and quantitation and reagent kit |
| CNB2004100399408A Expired - Fee Related CN1277934C (en) | 2000-06-08 | 2001-06-08 | Isometric primer extension method and kit for detection and quantification of specific nucleic acid |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011211156A Expired - Fee Related CN1198145C (en) | 2000-06-08 | 2001-06-08 | Isometric primer extension method for specific nucleate test and quantitation and reagent kit |
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| US (2) | US6824980B2 (en) |
| EP (1) | EP1162278B1 (en) |
| JP (1) | JP3789317B2 (en) |
| KR (1) | KR20010111020A (en) |
| CN (2) | CN1198145C (en) |
| CA (1) | CA2349810C (en) |
| DE (1) | DE60138242D1 (en) |
| HK (1) | HK1040769B (en) |
| RU (1) | RU2265058C2 (en) |
| SG (1) | SG153624A1 (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2001245698A (en) * | 1999-11-22 | 2001-09-11 | Xiao Bing Wang | Method for detecting nucleic acid |
| WO2004072294A2 (en) * | 2003-02-12 | 2004-08-26 | Genizon Svenska Ab | Methods and means for nucleic acid sequencing |
| WO2006128010A2 (en) * | 2005-05-26 | 2006-11-30 | The Trustees Of Boston University | Quantification of nucleic acids and proteins using oligonucleotide mass tags |
| US20070026430A1 (en) * | 2005-06-30 | 2007-02-01 | Applera Corporation | Proximity probing of target proteins comprising restriction and/or extension |
| JP2009545317A (en) * | 2006-08-01 | 2009-12-24 | アプライド バイオシステムズ， エルエルシー | Analyte and nucleic acid detection |
| WO2009123494A1 (en) * | 2008-04-01 | 2009-10-08 | Drygin Yury Fedorovich | Method for simultaneously detecting a plurality of rna sequences in a biological sample |
| CN106434873B (en) * | 2015-08-13 | 2021-08-27 | 生捷科技控股公司 | Method for synchronizing nucleic acid molecules |
| CN118048419A (en) * | 2017-10-04 | 2024-05-17 | 生捷科技控股公司 | Method and system for enzymatic synthesis of oligonucleotides |
| CN113166797B (en) | 2018-12-21 | 2024-04-12 | Illumina公司 | Nuclease-based RNA depletion |
| CN113557298A (en) * | 2019-03-13 | 2021-10-26 | 东洋纺株式会社 | Nucleic acid generation and amplification |
| KR20220035376A (en) * | 2019-06-18 | 2022-03-22 | 매머드 바이오사이언시즈 인크. | Assays and Methods for Nucleic Acid Detection |
| RU2748540C1 (en) * | 2021-02-08 | 2021-05-26 | Автономная некоммерческая образовательная организация высшего образования "Сколковский институт науки и технологий" | Method for detecting sars-cov-2 virus by mass spectrometry |
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| US4965188A (en) | 1986-08-22 | 1990-10-23 | Cetus Corporation | Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme |
| GB8805692D0 (en) | 1988-03-10 | 1988-04-07 | Ici Plc | Method of detecting nucleotide sequences |
| US5179198A (en) * | 1988-07-11 | 1993-01-12 | Hidechika Okada | Glycoprotein and gene coding therefor |
| US6013431A (en) | 1990-02-16 | 2000-01-11 | Molecular Tool, Inc. | Method for determining specific nucleotide variations by primer extension in the presence of mixture of labeled nucleotides and terminators |
| US5846710A (en) * | 1990-11-02 | 1998-12-08 | St. Louis University | Method for the detection of genetic diseases and gene sequence variations by single nucleotide primer extension |
| IE66125B1 (en) | 1991-01-31 | 1995-12-13 | Zeneca Ltd | Detection method for variant nucleotides |
| US5888819A (en) | 1991-03-05 | 1999-03-30 | Molecular Tool, Inc. | Method for determining nucleotide identity through primer extension |
| WO1993014217A1 (en) * | 1992-01-10 | 1993-07-22 | Life Technologies, Inc. | Use of predetermined nucleotides having altered base pairing characteristics in the amplification of nucleic acid molecules |
| US5710028A (en) | 1992-07-02 | 1998-01-20 | Eyal; Nurit | Method of quick screening and identification of specific DNA sequences by single nucleotide primer extension and kits therefor |
| US5650277A (en) * | 1992-07-02 | 1997-07-22 | Diagenetics Ltd. | Method of determining the presence and quantifying the number of di- and trinucleotide repeats |
| US6007987A (en) * | 1993-08-23 | 1999-12-28 | The Trustees Of Boston University | Positional sequencing by hybridization |
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| DE69432919T2 (en) | 1993-12-28 | 2004-05-27 | Tanabe Seiyaku Co., Ltd. | Methods for the detection of specific polynucleotides |
| AU5296496A (en) | 1995-03-24 | 1996-10-16 | Mitokor | Mutation detection by differential primer extension of mutant and wildtype target sequences |
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| US5830655A (en) * | 1995-05-22 | 1998-11-03 | Sri International | Oligonucleotide sizing using cleavable primers |
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| US7582420B2 (en) * | 2001-07-12 | 2009-09-01 | Illumina, Inc. | Multiplex nucleic acid reactions |
-
2001
- 2001-05-23 US US09/862,417 patent/US6824980B2/en not_active Expired - Lifetime
- 2001-06-01 JP JP2001166477A patent/JP3789317B2/en not_active Expired - Fee Related
- 2001-06-05 SG SG200103744-9A patent/SG153624A1/en unknown
- 2001-06-06 DE DE60138242T patent/DE60138242D1/en not_active Expired - Lifetime
- 2001-06-06 EP EP01304958A patent/EP1162278B1/en not_active Expired - Lifetime
- 2001-06-07 RU RU2001115828/13A patent/RU2265058C2/en not_active IP Right Cessation
- 2001-06-07 KR KR1020010031733A patent/KR20010111020A/en not_active Application Discontinuation
- 2001-06-07 CA CA2349810A patent/CA2349810C/en not_active Expired - Fee Related
- 2001-06-08 CN CNB011211156A patent/CN1198145C/en not_active Expired - Fee Related
- 2001-06-08 CN CNB2004100399408A patent/CN1277934C/en not_active Expired - Fee Related
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2002
- 2002-03-27 HK HK02102352.6A patent/HK1040769B/en not_active IP Right Cessation
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- 2004-01-14 US US10/757,263 patent/US20040137497A1/en not_active Abandoned
Also Published As
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| SG153624A1 (en) | 2009-07-29 |
| US20040137497A1 (en) | 2004-07-15 |
| CA2349810A1 (en) | 2001-12-08 |
| EP1162278B1 (en) | 2009-04-08 |
| CN1198145C (en) | 2005-04-20 |
| CA2349810C (en) | 2010-10-26 |
| EP1162278A3 (en) | 2004-04-14 |
| CN1537955A (en) | 2004-10-20 |
| US6824980B2 (en) | 2004-11-30 |
| DE60138242D1 (en) | 2009-05-20 |
| KR20010111020A (en) | 2001-12-15 |
| EP1162278A2 (en) | 2001-12-12 |
| US20030148525A1 (en) | 2003-08-07 |
| JP2002027993A (en) | 2002-01-29 |
| HK1040769A1 (en) | 2002-06-21 |
| CN1333465A (en) | 2002-01-30 |
| RU2265058C2 (en) | 2005-11-27 |
| HK1040769B (en) | 2005-08-12 |
| JP3789317B2 (en) | 2006-06-21 |
| RU2001115828A (en) | 2004-01-27 |
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